Hemopressin as a breakthrough for the cannabinoid field.

Hemopressin (PVNFKFLSH in rats, and PVNFKLLSH in humans and mice), a fragment derived from the α-chain of hemoglobin, was the first peptide described to have type 1 cannabinoid receptor activity. While hemopressin was shown to have inverse agonist/antagonistic activity, extended forms of hemopressin (i.e. RVD-hemopressin, also called pepcan-12) exhibit type 1 and type 2 cannabinoid receptor agonistic/allosteric activity, and recent studies suggest that they can activate intracellular mitochondrial cannabinoid receptors. Therefore, hemopressin and hemopressin-related peptides could have location-specific and biased pharmacological action, which would increase the possibilities for fine-tunning and broadening cannabinoid receptor signal transduction. Consistent with this, hemopressins were shown to play a role in a number of physiological processes including antinociceptive and anti-inflammatory activity, regulation of food intake, learning and memory. The shortest active hemopressin fragment, NFKF, delays the first seizure induced by pilocarpine, and prevents neurodegeneration in an experimental model of autoimmune encephalomyelitis. These functions of hemopressins could be due to engagement of both cannabinoid and non-cannabinoid receptor systems. Self-assembled nanofibrils of hemopressin have pH-sensitive switchable surface-active properties, and show potential as inflammation and cancer targeted drug-delivery systems. Upon disruption of the self-assembled hemopressin nanofibril emulsion, the intrinsic analgesic and anti-inflammatory properties of hemopressin could help bolster the therapeutic effect of anti-inflammatory or anti-cancer formulations. In this article, we briefly review the molecular and behavioral pharmacological properties of hemopressins, and summarize studies on the intricate and unique mode of generation and binding of these peptides to cannabinoid receptors. Thus, the review provides a window into the current status of hemopressins in expanding the repertoire of signaling and activity by the endocannabinoid system, in addition to their new potential for pharmaceutic formulations.

Hemopressin as a breakthrough for the cannabinoid field

Hemopressin (PVNFKFLSH in rats, and PVNFKLLSH in humans and mice), a fragment derived from the α-chain of hemoglobin, was the first peptide described to have type 1 cannabinoid receptor activity. While hemopressin was shown to have inverse agonist/antagonistic activity, extended forms of hemopressin (i.e. RVD-hemopressin, also called pepcan-12) exhibit type 1 and type 2 cannabinoid receptor agonistic/allosteric activity, and recent studies suggest that they can activate intracellular mitochondrial cannabinoid receptors. Therefore, hemopressin and hemopressin-related peptides could have location-specific and biased pharmacological action, which would increase the possibilities for fine-tunning and broadening cannabinoid receptor signal transduction. Consistent with this, hemopressins were shown to play a role in a number of physiological processes including antinociceptive and anti-inflammatory activity, regulation of food intake, learning and memory. The shortest active hemopressin fragment, NFKF, delays the first seizure induced by pilocarpine, and prevents neurodegeneration in an experimental model of autoimmune encephalomyelitis. These functions of hemopressins could be due to engagement of both cannabinoid and non-cannabinoid receptor systems. Self-assembled nanofibrils of hemopressin have pH-sensitive switchable surface-active properties, and show potential as inflammation and cancer targeted drug-delivery systems. Upon disruption of the self-assembled hemopressin nanofibril emulsion, the intrinsic analgesic and anti-inflammatory properties of hemopressin could help bolster the therapeutic effect of anti-inflammatory or anti-cancer formulations. In this article, we briefly review the molecular and behavioral pharmacological properties of hemopressins, and summarize studies on the intricate and unique mode of generation and binding of these peptides to cannabinoid receptors. Thus, the review provides a window into the current status of hemopressins in expanding the repertoire of signaling and activity by the endocannabinoid system, in addition to their new potential for pharmaceutic formulations.

Thimet Oligopeptidase (EC 3.4.24.15) Key Functions Suggested by Knockout Mice Phenotype Characterization.

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Thimet oligopeptidase (ec 3.4.24.15) key functions suggested by knockout mice phenotype characterization

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Thimet oligopeptidase (ec 3.4.24.15) key functions suggested by knockout mice phenotype characterization

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Substrate Capture Assay Using Inactive Oligopeptidases to Identify Novel Peptides.

Researchers are always searching for novel biologically active molecules including peptides. With the improvement of equipment for electrospray mass spectrometry, it is now possible to identify hundreds of novel peptides in a single run. However, after identifying the peptide sequences it is expensive to synthesize all the peptides to perform biological activity assays. Here, we describe a substrate capture assay that uses inactive oligopeptidases to identify putative biologically active peptides in complexes peptide mixtures. This methodology can use any crude extracts of biological tissues or cells, with the advantage to introduce a filter (i.e., binding to an inactive oligopeptidase) as a prior step in screening to bioactive peptides.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome.

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251-259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo, when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome ß5i subunit and to increase the proliferation of CD8+ T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity. BIOLOGICAL SIGNIFICANCE: The functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.

Peptidomic analysis of the anterior temporal lobe and corpus callosum from schizophrenia patients

Schizophrenia is a complex disorder hypothesized to develop from a combination of genetic, neurodevelopmental, and environmental factors. Molecules that are directly involved in the pathogenesis of schizophrenia and may serve as biomarker candidates can be identified with "omics" approaches such as proteomics and peptidomics. In this context, we performed a peptidomic study in schizophrenia postmortem brains, to our knowledge the first such study in schizophrenia patients. We investigated the anterior temporal lobe (ATL) and corpus callosum (CC) by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and a label-free ion quantification technique based on data-dependent acquisition (DDA). Results indicated alterations in a specific intracellular neurogranin peptide in both the ATL and CC and a decrease of PepH, a fragment of his tone H2B type 1-H intracellular peptide, in the ATL. PepH was tested in serum-deprived Neuro2A cells and showed a protective effect against cell death. Cells were also challenged with lipopolysaccharide (LPS), and PepH was able to prevent the endotoxic effects of LPS. Our data suggest that specific intracellular peptides are altered in schizophrenia patients. The potential biological activity of PepH supports intracellular peptides as novel targets in the study not only of schizophrenia but also of other neuropsychiatric diseases. Biological significance: Psychiatric disorders are considerably more difficult to diagnose in their early stages. Usually, by the time the diagnosis is clear and clinical treatment can be started, the disorder is already established and thus of greater severity. Consequently, the scientific community has been searching for biomarker candidates that can aid the early detection of such disorders and for novel therapeutics to improve treatment or at least delay disease progression. Moreover, key molecules involved in the establishment of psychiatric diseases may help the understanding of their pathogenesis and thus drive the development of more effective treatments. The present work screened peptides that might be possible novel targets to control cell machinery in schizophrenia and identified an intracellular peptide with potential cytoprotective activity. To our knowledge, this is the first peptidomic study in schizophrenia patients.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251-259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo, when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome beta 5i subunit and to increase the proliferation of CD8 + T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity.Biological significance: The functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.

[des-Arg(1)]-Proctolin: A novel NEP-like enzyme inhibitor identified in Tityus serrulatus venom.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

Anxiogenic-like effects induced by hemopressin in rats.

Hemopressin (PVNFKFLSH; HP) is an orally active peptide derived from rat hemoglobin α-chain that could act as an inverse agonist at cannabinoid type 1 receptors (CB1). Here, we aim to investigate possible behavioral effects of HP in male Wistar rats tested in the elevated plus maze (EPM), following HP intraperitoneal (i.p., 0.05 mg/kg), oral (P.O., 0.05 and 0.5 mg/kg) or intracerebroventricular (I.C.V., 3 and 10 nmol) administration. HP induced a decrease in EPM open arm exploration, indicating an anxiogenic-like effect. However, i.p. administration of HP (1 mg/kg) followed by mass spectrometry analysis of brain-peptide extracts suggested that the intact HP does not cross the blood brain barrier. I.C.V. administrated HP produced anxiogenic-like effects that were prevented by Transient Receptor Potential Vanilloid Type 1 (TRPV1) antagonists, 6-iodonordihydrocapsaicin (1 nmol) or SB366791 (1 nmol), but not by the CB1 receptor antagonist AM251 (0.1 and 1 nmol). Altogether, these data suggest that I.C.V. administrated HP induces anxiogenic-like effects by activating TRPV1 receptors. The similar anxiogenic effects observed after i.p. or P.O. administration could be due to HP fragment(s) crossing the blood brain barrier. The present results advance our knowledge about HP pharmacology and suggest concerns in future clinical studies.

Anxiogenic-like effects induced by hemopressin in rats

Hemopressin (PVNFKFLSH; HP) is an orally active peptide derived from rat hemoglobin alpha-chain that could act as an inverse agonist at cannabinoid type 1 receptors (CB1). Here, we aim to investigate possible behavioral effects of HP in male Wistar rats tested in the elevated plus maze (EPM), following HP intraperitoneal (i.p., 0.05 mg/kg), oral (P.O., 0.05 and 0.5 mg/kg) or intracerebroventricular (I.C.V., 3 and 10 nmol) administration. HP induced a decrease in EPM open arm exploration, indicating an anxiogenic-like effect. However, i.p. administration of HP (1 mg/kg) followed by mass spectrometry analysis of brain-peptide extracts suggested that the intact HP does not cross the blood brain barrier. I.C.V. administrated HP produced anxiogenic-like effects that were prevented by Transient Receptor Potential Vanilloid Type 1 (TRPV1) antagonists, 6-iodonordihydrocapsaicin (1 nmol) or SB366791 (1 nmol), but not by the CB1 receptor antagonist AM2S1 (0.1 and 1 nmol). Altogether, these data suggest that I.C.V. administrated HP induces anxiogenic-like effects by activating TRPV1 receptors. The similar anxiogenic effects observed after i.p. or P.O. administration could be due to HP fragment(s) crossing the blood brain barrier. The present results advance our knowledge about HP pharmacology and suggest concerns in future clinical studies.

Peptidomic analysis of the neurolysin-knockout mouse brain.

A large number of intracellular peptides are constantly produced following protein degradation by the proteasome. A few of these peptides function in cell signaling and regulate protein-protein interactions. Neurolysin (Nln) is a structurally defined and biochemically well-characterized endooligopeptidase, and its subcellular distribution and biological activity in the vertebrate brain have been previously investigated. However, the contribution of Nln to peptide metabolism in vivo is poorly understood. In this study, we used quantitative mass spectrometry to investigate the brain peptidome of Nln-knockout mice. An additional in vitro digestion assay with recombinant Nln was also performed to confirm the identification of the substrates and/or products of Nln. Altogether, the data presented suggest that Nln is a key enzyme in the in vivo degradation of only a few peptides derived from proenkephalin, such as Met-enkephalin and octapeptide. Nln was found to have only a minor contribution to the intracellular peptide metabolism in the entire mouse brain. However, further studies appear necessary to investigate the contribution of Nln to the peptide metabolism in specific areas of the murine brain. BIOLOGICAL SIGNIFICANCE: Neurolysin was first identified in the synaptic membranes of the rat brain in the middle 80's by Frederic Checler and colleagues. Neurolysin was well characterized biochemically, and its brain distribution has been confirmed by immunohistochemical methods. The neurolysin contribution to the central and peripheral neurotensin-mediated functions in vivo has been delineated through inhibitor-based pharmacological approaches, but its genuine contribution to the physiological inactivation of neuropeptides remains to be firmly established. As a result, the main significance of this work is the first characterization of the brain peptidome of the neurolysin-knockout mouse. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Guest Editors: César López-Camarillo, Victoria Pando-Robles and Bronwyn Jane Barkla.

A novel intracellular peptide derived from g1/s cyclin d2 induces cell death.

Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25-100 µm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity.

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Insights into scorpion venom peptides: alternative processing of ß-KTx propeptide from Tityus serrulatus venom results in a new naturally occurring thimet oligopeptidase inhibitor.

Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules on ion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the ß-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms.

Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes.

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.